The primary functions of the gastrointestinal tract traditionally include digestion and absorption of nutrients, and electrolytes and water homeostasis. Recent studies indicate that another key function of this organ is to regulate trafficking of environmental antigens across the host mucosal barrier. Together with the gut- associated lymphoid tissue and the neuroendocrine network, the intestinal epithelial barrier, with its intercellular tight junctions (TJ), controls the balance between tolerance and immunity to non-self antigens. During the continuous 15 years of funding of this grant, we have used a Vibrio cholerae-elaborated protein, zonula occludens toxin (Zot) as a tool to gain insights into the regulation of TJ function. Our studies led to the discovery of zonulin, a human eukaryotic Zot homologue, and to the definition of aspects of its physiological and pathological functions. To our knowledge, zonulin is the only reversible modulator of intercellular TJs described so far. Genome wide association studies have coupled genes controlling gut permeability to autoimmune and inflammatory diseases. Our studies have demonstrated that zonulin expression is increased in autoimmune disorders associated with gut barrier dysfunction, such as celiac disease and type 1 diabetes. Our data generated on intestinal cell lines, animal models, and human tissues indicate that Zot and zonulin both induce proteinase-activated receptor 2 (PAR2)-mediated transactivation of epidermal growth factor receptor (EGFR). Our overall hypothesis is that this PAR2-mediated transactivation of EGFR is coupled with TJ disassembly, and, ultimately, increased intestinal permeability. Further we hypothesize that the number of zonulin gene copies is associated with augmented zonulin expression and a constitutively dysfunctional, permissive gut mucosal barrier. To challenge these hypotheses, we propose three specific aims. With Aim 1 we will expand our studies to establish the mechanisms through which zonulin because PAR2- mediated transactivation of EGF. We have demonstrated that this transactivation is followed by protein kinase C (PKC)a-mediated ZO-1 and myosin 1c serine/threonine phosphorylation, provoking ZO-1 disengagement from three of its binding partners, occludin, claudin-1, and transiently from myosin 1C, and induce actin polymerization, ultimately causing TJ disassembly. With Aim 2 we will define the zonulin-triggered intracellular signaling events downstream to EGFR activation leading to increased intestinal permeability. We have recently established identity between zonulin and pre-haptoglobin (HP) 2, i.e., the uncleaved product of HP2. The HP2- 2 genotype is more frequently encountered in immune-mediated conditions, including celiac disease, Crohn's disease, and schizophrenia, and its expression is associated with more severe clinical manifestations. With our Aim 3 we will investigate the association of HP genotypes (HP1-1, HP1-2, and HP2-2) with serum zonulin expression and clinical severity in human disease states and transgenic murine models.